Abstract: Fluorescent biosensor techniques in cell biology now allow for the real-time interrogation of molecular processes as they occur inside living cells at spatial and temporal resolutions of microns and seconds, respectively. I have a prior and continued focus on the development of novel Frster resonance energy transfer (FRET)- based biosensor technologies that utilize monomeric fluorescent proteins for exceptional sensitivity and probe reversibility. In addition, a single-chain construction is used to facilitate quantitative analysis. I recently pioneered the near-infrared (NIR)-FRET biosensor modality, which included the first simultaneous, orthogonal visualization of cyan-yellow fluorescent protein-based FRET and NIR-FRET biosensors in single living cells. The resulting data were the first true multiplex analysis of two important molecular switches in living cells, the Rac1 and RhoA GTPases. This analysis revealed the direct coordination of these GTPases during cell migration in real-time. Herein, I propose to study the coordination of Rho GTPases associated with important signaling pathways by designing new biosensors for specific signaling nodes and utilizing the direct multiplex FRET imaging approach. Specifically, I will first target the local-level coordination of Rho GTPase signaling in fibroblast cells during migration, chemotaxis, and directional guidance. The coordination of RhoA versus Rac1 GTPases in fibroblasts will be investigated by determining the role of a downstream target protein, the formin mDia1, which is hypothesized to coordinate RhoA and Rac1 during cell motility. The direct multiplex imaging approach will be used to evaluate pairwise biosensor signals. In addition, the RhoA and Rac1 pathways will be perturbed with optogenetic tools to determine the GTPase coordination that is important for controlling cellular morphodynamics. Next, these approaches will be applied to two systems that have important implications for human health and disease. First, macrophage motility and directional guidance, which requires the coordination of Rho GTPases during the chemotactic response to inflammatory chemokines, will be studied. Then, the multiplex imaging and perturbation approaches will be applied to breast cancer invasion and migration, which are critical to controlling tumor metastasis. Collectively, the coordination between Rho GTPases and the associated molecular signaling that governs cell motility will be identified through the development of new biosensors that enable direct multiplex probing of signaling networks.